The nodes of Ranvier of myelinated axons are critical for action potential propagation by saltatory conduction and are a striking example of the exquisite domain organization characteristic of neurons. Nodes are comprised of a multimeric complex that includes voltage gated sodium and potassium channels, accessory beta subunits, cell adhesion molecules (CAMs), and a cytoskeletal complex that includes ankyrin G to which channels and CAMs bind. Our recent studies indicate that the node assembles from distinct sources. CAMs, notably NF186, accumulate at forming nodes by redistribution from existing pools on the axon surface via diffusion trapping from interactions with the Schwann cell. In contrast, channels and cytoskeletal proteins are delivered to the node primarily via axonal transport. Surprisingly, there also appears to be a pool of sodium channels that traffics and accumulates at the node independent of transport and of ankyrin G. Finally, our studies suggest there is an active program to clear nodal proteins from extranodal sites that further reinforces their selective enrichment at the node. To extend these findings and further elucidate the mechanisms of node assembly, we will investigate trafficking and assembly of components of the node, and how they are cleared from extranodal sites, by live imaging strategies in myelinating cocultures. In particular, we will: i) determine whether vesicles that transport components to the nodes segregate into those specific for CAMs and others for ion channels, and exhibit any domain specificity, ii) characterize transport-dependent and -independent trafficking of sodium channel components to the node, including the roles of ankyrin G and NF186, respectively, and determine whether the sodium channel complex assembles locally, and i) investigate how nodal components are cleared from extranodal sites, focusing on NF186 to examine further the role of its cytoplasmic segment and that of endocytosis and proteolysis in clearing the internodal, surface pool of NF186. Relevance: These studies will elucidate the mechanisms that regulate the assembly of the node of Ranvier, which is critical to the ability of nerve fibers to conduct electrical impulses appropriately. Findings in this study may therefore have important implications for our understanding of the pathogenesis of disorders of myelinated fibers, including neuropathies that result in aberrant nerve conduction, and may thereby lead to new therapeutic strategies for these neurological disorders.